Distinct organization of two cortico-cortical feedback pathways

Neocortical feedback is critical for attention, prediction, and learning. To mechanically understand its function requires deciphering its cell-type wiring. Recent studies revealed that feedback between primary motor to primary somatosensory areas in mice is disinhibitory, targeting vasoactive intestinal peptide-expressing interneurons, in addition to pyramidal cells. It is unknown whether this circuit motif represents a general cortico-cortical feedback organizing principle. Here we show that in contrast to this wiring rule, feedback between higher-order lateromedial visual area to primary visual cortex preferentially activates somatostatin-expressing interneurons. Functionally, both feedback circuits temporally sharpen feed-forward excitation eliciting a transient increase–followed by a prolonged decrease–in pyramidal cell activity under sustained feed-forward input. However, under feed-forward transient input, the primary motor to primary somatosensory cortex feedback facilitates bursting while lateromedial area to primary visual cortex feedback increases time precision. Our findings argue for multiple cortico-cortical feedback motifs implementing different dynamic non-linear operations.

In a subset of experiments, we recovered the morphologies of the recorded cells to confirm their cell classes. Morphologies and firing patterns were particularly important in two cases in our experiments. First, it has been reported in previous studies 1-3 that a subset of SOM+ cells are fast-spiking (FS) basket cells (Fig S2a and b, bottom), while the rest of the cells are non-fast-spiking (Non-FS) cells (Fig S2a and  b, top). As the two subsets of cells are very different in the morphology and firing pattern (Fig S2a and b), we excluded the activities of FS cells in the main analyses, although their log2 normalized EPSPs are not significantly different (Fig S2c and d). Second, in the bursting experiments (Fig 5), we mainly used the firing pattern to identify the intrinsic bursty cells in L5 in V1 or vS1. The firing pattern of the bursty cells was featured with their high firing rate (> 100 Hz, Fig S3) at the beginning of the current injection and the large afterhyperpolarization (AHP) following the bursting. We morphologically recovered a subset of the cells with bursty firing patterns and consistent with previous literature, these cells were thick-tufted cells ( Fig S3).  Intrinsic bursty cells and Non-bursty cells in L5 were identified with both the morphologies and the firing patterns. a Morphologies and firing patterns of example thick-tufted bursty cell (left, blue) and thin-tufted non-bursty cell (right, black) in V1. b Morphologies and firing patterns of example thick-tufted bursty cell (left, blue) and thin-tufted non-bursty cell (right, black) in vS1. Source data are provided as a Source Data file.

Blue LED stimulation do not elicit activities on cells without ChR2
To exclude the possibility that blue LED stimulation itself elicits electrical responses, we recorded ESPCs four cells in the visual cortex in absence of ChR2 when stimulated with 2 ms LED pulses. We found no ESPCs in these cells in response to the LED simulation ( Fig S4).

Most of the recorded EPSC and EPSP events were monosynaptic
To identify whether the elicited events were monosynaptic, in a subset of the connectivity experiments, we applied the sodium channel blocker tetrodotoxin (TTX, 1 µM) and the potassium channel blocker 4-aminopyridine (4-AP, 500 µM) 4 . TTX blocks action potentials to prevent polysynaptic events, while 4-AP enhances ChR2-mediated depolarization of axonal boutons, increasing the detectability of monosynaptic events 4,5 . We found that all light-evoked synaptic events were silenced by TTX alone, but most of them could be partially recovered by the addition of 4-AP (Fig S5a and b), indicating that most of the excitatory responses we recorded were indeed monosynaptic. We selected 7 ms as a latency cutoff for responsive cells in all subsequent experiments, because we found that all cells that were recoverable by the addition of 4-AP had latencies less than 7 ms (Fig S5c). Although there are cells with latencies smaller than 7 ms that are not recoverable (5/43, Fig S5c), we decided not to use a shorter latency as the cutoff because of the following reasons. First, the TTX-4-AP approach is a stringent test for monosynaptic events. That is, it is almost impossible for polysynaptic responses to recover, while some small monosynaptic events may fail to recover because the presence of TTX-4-AP typically causes a significant reduction in the EPSC amplitudes 4,5 . Second, the latencies in our study are longer than typical latencies in other literature 6 . This is because our recordings were performed under room temperature instead of physiological temperature, which preserves the quality of tissues 7,8 . It is possible that some events by this standard were polysynaptic, but we found that the recorded excitatory cells were never driven to fire by feedback activation (Fig 3e), indicating a low rate of polysynaptic events. We have also used 4 ms as the latency cutoff and redid the analysis ( Fig S6) and our main conclusions preserved.

Fig S5.
Identify monosynaptic events with pharmacological methods. a Example traces of feedback (blue ticks) responses with no drug (black), TTX (orange), and both TTX and 4-AP (green). The cells that were still responsive in the presence of TTX and 4-AP were identified as recoverable cells. b Proportion of cells that are non-responsive without drug (gray), recoverable (green) and unrecoverable (orange) in V1 (left) or vS1 (right). c Distribution of the average latencies across traces for recoverable cells and unrecoverable cells. We set 7 ms as the latency cutoff for presumably monosynaptic events. Source data are provided as a Source Data file.

Raw values of EPSC and EPSP show that vM1 to vS1 feedback pathway might have an overall weaker connection than LM to V1 pathway
In the main text (Fig 3), we have normalized the EPSC and EPSP amplitudes to those of L2/3 pyramidal cells to account for the variability in the amount of virus injection across the animals. In Fig S7, we show the raw values of EPSC and EPSP. Under the assumption that the strength and amount of virus injected were similar among animals, we found that EPSC and EPSP of most of the cell classes in the vM1 to vS1 pathway were significantly lower than those in LM to V1 (Fig S7, Table S5), suggesting an overall weaker connection of vM1 to vS1 feedback pathway. Note that the raw EPSC values in VIP+ cells were similar between V1 and vS1, but raw EPSP values were higher in vS1 than in V1 (p = 0.004, Mann Whitney U test, p values adjusted with Benjamin-Hochberg method). This is because the input resistance of VIP+ cells in vS1 (median 287 MΩ) is higher than that of VIP+ cells in V1 (median 204 MΩ, Fig S8,

Variability of "temporal sharpening" effect across cells
In Figs S9 and S10, we show more examples of the "temporal sharpening effect". While the temporal sharpening of L2/3 and L5 pyramidal cells was highly consistent across cells (Fig S9 and Fig S10, a and  c), the effect on excitatory cells in L4 was variable (Fig S9b and Fig S10b).

Comparison of burstiness for L5 IB neurons in V1 and vS1
A previous study showed differences in dendritic integration properties of L5 cells along the rostral-caudal axis in the cortex, and neurons in S1 are likely to be more prone to bursting than those cells in V1 9 . To account for the difference in the population of L5 pyramidal cells, we preselected bursty neurons to perform the feedback stimulation protocols. These neurons, as reported in the literature, are thick-tufted neurons in both V1 10 and vS1 11 . To directly compare their burstiness in our experiments, we recorded the firing patterns of the neurons and measured how prone they were to burst with two metrics: minimal current to elicit a burst and burstiness. Burstiness was defined as the firing rate difference of the spikes inside bursts from that out of the bursts, normalized by their summation (see Methods). We found that there were no significant differences between V1 bursty neurons and vS1 bursty neurons in both metrics (Fig S11), indicating that the difference we found in bursting with feedback stimulation is not a result of the difference in their intrinsic bursting properties.

Comparison of light-evoked EPSCs of V1 and vS1 neurons in the bursting experiments
In the bursting experiments, to make the results comparable across experiments, we recorded the light-evoked EPSCs before the bursting protocol and adjusted the LED light intensity to elicit EPSC ranging from 100 pA to 500 pA. We showed in Fig S12 that there was no significant difference in EPSC between cells in V1 and vS1 (Fig S12).

Fig S12.
Light-evoked EPSCs of V1 and vS1 intrinsic bursty neurons. The bars mark the median values. p = 0.55, two-sided Wilcoxon rank-sum test. V1: 11 cells from 2 animals. S1: 15 cells from 5 animals. Source data are provided as a Source Data file.

Rise time, decay time and time to peak
To identify whether the difference we found in feedback-evoked bursting was because of the difference in the excitatory response of L5 cells, we measured the rise time, decay time and time to peak for feedback eliciting EPSC in L5 pyramidal cells, but found no significant difference in those measures ( Fig S13). This result indicates the difference in the bursting was more likely due to the difference in feedback-evoked inhibitory circuits.